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Silencing of CD47 and SIRP α by Polypurine reverse Hoogsteen hairpins to promote MCF-7 breast cancer cells death by PMA-differentiated THP-1 cells

View Article: PubMed Central - PubMed

ABSTRACT

Background: In the context of tumor immunology, tumor cells have been shown to overexpress CD47, an anti-phagocytic signal directed to macrophages to escape from phagocytosis by interacting with Signal Regulatory Protein α SIRPα.

Background: In the present work, we designed Polypurine reverse Hoogsteen hairpins, PPRHs, to silence the expression of CD47 in tumor cells and SIRPα in macrophages with the aim to eliminate tumor cells by macrophages in co-culture experiments.

Methods: THP-1 cells were differentiated to macrophages with PMA. The mRNA levels of differentiation markers CD14 and Mcl-1 mRNA and pro-inflammatory cytokines (IL-1β, IL-18, IL-6, IL-8 and TNF-α) were measured by qRT-PCR. The ability of PPRHs to silence CD47 and SIRPα was evaluated at the mRNA level by qRT-PCR and at the protein level by Western Blot. Macrophages were co-cultured with tumor cells in the presence of PPRHs to silence CD47 and/or SIRPα. Cell viability was assessed by MTT assays.

Results: THP-1 cells differentiated to macrophages with PMA showed an increase in macrophage surface markers (CD14, Mcl-1) and pro-inflammatory cytokines (IL-1β, IL-18, IL-6, IL-8 and TNF-α). PPRHs were able to decrease both CD47 expression in MCF-7 cell line and SIRPα expression in macrophages at the mRNA and protein levels. In the presence of PPRHs, MCF-7 cells were eliminated by macrophages in co-culture experiments, whereas they survived in the absence of PPRHs.

Conclusions: Our data support the usage of PPRHs to diminish CD47/SIRPα interaction by decreasing the expression of both molecules thus resulting in an enhanced killing of MCF-7 cells by macrophages, which might translate into beneficial effects in cancer therapy. These results indicate that PPRHs could represent a new approach with immunotherapeutic applications.

No MeSH data available.


Co-culture experiments. In the co-culture experiments, either only MCF-7 cells (60,000) were transfected with HpCD47Pr-T, only THP-1 cells (1000) with HpSIRPαI7-T or both MCF-7 and THP-1 cells were transfected with the corresponding PPRH or scrambled PPRHs in controls. As positive control an antibody anti-CD47 was used. Then THP-1 cells were differentiated with 3 ng/ml PMA. The percentage of viable cells was calculated relative to the control of MCF-7 cells incubated with 3 ng/ml PMA. Data represent the mean ± SE of at least three experiments (*p < 0.05, **p < 0.01, ***p < 0.005)
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Fig5: Co-culture experiments. In the co-culture experiments, either only MCF-7 cells (60,000) were transfected with HpCD47Pr-T, only THP-1 cells (1000) with HpSIRPαI7-T or both MCF-7 and THP-1 cells were transfected with the corresponding PPRH or scrambled PPRHs in controls. As positive control an antibody anti-CD47 was used. Then THP-1 cells were differentiated with 3 ng/ml PMA. The percentage of viable cells was calculated relative to the control of MCF-7 cells incubated with 3 ng/ml PMA. Data represent the mean ± SE of at least three experiments (*p < 0.05, **p < 0.01, ***p < 0.005)

Mentions: Based on the patterns of pro-inflammatory cytokines and surface markers levels, 3 ng/ml of PMA was chosen for THP-1 differentiation. To decrease CD47/SIRPα interaction, CD47 and SIRPα alone or in combination were targeted by PPRHs. Scrambled PPRHs were used as negative controls and an antibody anti-CD47 as positive control to disrupt the interaction CD47/SIRPα (Fig. 5). By decreasing the level of CD47 in tumor cells, 60 % of MCF-7 cells were killed by macrophages (Fig. 5). When transfecting THP-1 cells with HpSIRPαI7-T, the decreased level of SIRPα allowed macrophages to kill 58 % of tumor cells (Fig. 5). To decrease the level of both targets in the same co-culture and to better kill tumor cells, THP-1 cells were first transfected with HpSIRPαI7-T and after differentiation, MCF-7 cells were transfected with HpCD47Pr-T in the co-culture. In these conditions, 70 % of tumor cells were eliminated. Similar results were obtained under anti-CD47 treatment. On the other hand, tumor cells escaped from macrophage killing in the absence of PPRHs and the cell viability when transfected with scrambled PPRHs was reduced by 25 % compared to that of the co-culture control (Fig. 5).Fig. 5


Silencing of CD47 and SIRP α by Polypurine reverse Hoogsteen hairpins to promote MCF-7 breast cancer cells death by PMA-differentiated THP-1 cells
Co-culture experiments. In the co-culture experiments, either only MCF-7 cells (60,000) were transfected with HpCD47Pr-T, only THP-1 cells (1000) with HpSIRPαI7-T or both MCF-7 and THP-1 cells were transfected with the corresponding PPRH or scrambled PPRHs in controls. As positive control an antibody anti-CD47 was used. Then THP-1 cells were differentiated with 3 ng/ml PMA. The percentage of viable cells was calculated relative to the control of MCF-7 cells incubated with 3 ng/ml PMA. Data represent the mean ± SE of at least three experiments (*p < 0.05, **p < 0.01, ***p < 0.005)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037635&req=5

Fig5: Co-culture experiments. In the co-culture experiments, either only MCF-7 cells (60,000) were transfected with HpCD47Pr-T, only THP-1 cells (1000) with HpSIRPαI7-T or both MCF-7 and THP-1 cells were transfected with the corresponding PPRH or scrambled PPRHs in controls. As positive control an antibody anti-CD47 was used. Then THP-1 cells were differentiated with 3 ng/ml PMA. The percentage of viable cells was calculated relative to the control of MCF-7 cells incubated with 3 ng/ml PMA. Data represent the mean ± SE of at least three experiments (*p < 0.05, **p < 0.01, ***p < 0.005)
Mentions: Based on the patterns of pro-inflammatory cytokines and surface markers levels, 3 ng/ml of PMA was chosen for THP-1 differentiation. To decrease CD47/SIRPα interaction, CD47 and SIRPα alone or in combination were targeted by PPRHs. Scrambled PPRHs were used as negative controls and an antibody anti-CD47 as positive control to disrupt the interaction CD47/SIRPα (Fig. 5). By decreasing the level of CD47 in tumor cells, 60 % of MCF-7 cells were killed by macrophages (Fig. 5). When transfecting THP-1 cells with HpSIRPαI7-T, the decreased level of SIRPα allowed macrophages to kill 58 % of tumor cells (Fig. 5). To decrease the level of both targets in the same co-culture and to better kill tumor cells, THP-1 cells were first transfected with HpSIRPαI7-T and after differentiation, MCF-7 cells were transfected with HpCD47Pr-T in the co-culture. In these conditions, 70 % of tumor cells were eliminated. Similar results were obtained under anti-CD47 treatment. On the other hand, tumor cells escaped from macrophage killing in the absence of PPRHs and the cell viability when transfected with scrambled PPRHs was reduced by 25 % compared to that of the co-culture control (Fig. 5).Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Background: In the context of tumor immunology, tumor cells have been shown to overexpress CD47, an anti-phagocytic signal directed to macrophages to escape from phagocytosis by interacting with Signal Regulatory Protein &alpha; SIRP&alpha;.

Background: In the present work, we designed Polypurine reverse Hoogsteen hairpins, PPRHs, to silence the expression of CD47 in tumor cells and SIRP&alpha; in macrophages with the aim to eliminate tumor cells by macrophages in co-culture experiments.

Methods: THP-1 cells were differentiated to macrophages with PMA. The mRNA levels of differentiation markers CD14 and Mcl-1 mRNA and pro-inflammatory cytokines (IL-1&beta;, IL-18, IL-6, IL-8 and TNF-&alpha;) were measured by qRT-PCR. The ability of PPRHs to silence CD47 and SIRP&alpha; was evaluated at the mRNA level by qRT-PCR and at the protein level by Western Blot. Macrophages were co-cultured with tumor cells in the presence of PPRHs to silence CD47 and/or SIRP&alpha;. Cell viability was assessed by MTT assays.

Results: THP-1 cells differentiated to macrophages with PMA showed an increase in macrophage surface markers (CD14, Mcl-1) and pro-inflammatory cytokines (IL-1&beta;, IL-18, IL-6, IL-8 and TNF-&alpha;). PPRHs were able to decrease both CD47 expression in MCF-7 cell line and SIRP&alpha; expression in macrophages at the mRNA and protein levels. In the presence of PPRHs, MCF-7 cells were eliminated by macrophages in co-culture experiments, whereas they survived in the absence of PPRHs.

Conclusions: Our data support the usage of PPRHs to diminish CD47/SIRP&alpha; interaction by decreasing the expression of both molecules thus resulting in an enhanced killing of MCF-7 cells by macrophages, which might translate into beneficial effects in cancer therapy. These results indicate that PPRHs could represent a new approach with immunotherapeutic applications.

No MeSH data available.